FRAP and FRET methods to study nuclear receptors in living cells.

Quantitative imaging techniques of fluorescently-tagged proteins have been instrumental in the study of the behavior of nuclear receptors (NRs) and coregulators in living cells. Ligand-activated NRs exert their function in transcription regulation by binding to specific response elements in promotor and enhancer sequences of genes. Fluorescence recovery after photobleaching (FRAP) has proven to be a powerful tool to study the mobility of fluorescently-labeled molecules in living cells. Since binding to DNA leads to the immobilization of DNA-interacting proteins like NRs, FRAP is especially useful for determining DNA-binding kinetics of these proteins. The coordinated interaction of NRs with promoters/enhancers and subsequent transcription activation is not only regulated by ligand but also by interactions with sets of cofactors and, at least in the case of the androgen receptor (AR), by dimerization and interdomain interactions. In living cells, these interactions can be studied by fluorescence resonance energy transfer (FRET). Here we provide and discuss detailed protocols for FRAP and FRET procedures to study the behavior of nuclear receptors in living cells. On the basis of our studies of the AR, we provide protocols for two different FRAP methods (strip-FRAP and FLIP-FRAP) to quantitatively investigate DNA-interactions and for two different FRET approaches, ratio imaging, and acceptor photobleaching FRET to study AR domain interactions and interactions with cofactor motifs. Finally, we provide a protocol of a technique where FRAP and acceptor photobleaching FRET are combined to study the dynamics of interacting ARs.

Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells.

Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.

Antiandrogens prevent stable DNA-binding of the androgen receptor.

The androgen receptor (AR) is essential for development of the male gender and in the growth of the majority of prostate cancers. Agonists as well as most antagonists induce translocation of the receptor to the nucleus, whereas only agonists can activate AR function. Antagonists are therefore used in the therapy of metastasized prostate cancer. To obtain insight into the mechanism by which antagonists block AR function in living cells, we studied nuclear mobility and localization of green fluorescent protein (GFP)-tagged AR in the presence of either the agonist R1881 or the antagonists bicalutamide and hydroxyflutamide. As controls we investigated a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with broadened ligand specificity. We demonstrate that in the presence of R1881, AR localizes in numerous intranuclear foci and, using complementary fluorescence recovery after photobleaching (FRAP) approaches and computer modelling, that a fraction of AR ( approximately 10-15%) is transiently immobilized in a DNA-binding-dependent manner (individual ARs being immobile for approximately 45 seconds). By contrast, antagonist-bound GFP-AR showed no detectable immobile fraction and the mobility was similar to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating that antagonists do not induce the relatively stable DNA-binding-dependent immobilization observed with agonist-bound AR. Moreover, in the presence of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed in the nucleus. Binding of bicalutamide and hydroxyflutamide to GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar mobility and heterogeneous nuclear distribution as observed for R1881-liganded GFP-AR. The live cell studies indicate that the investigated antagonists interfere with events early in the transactivation function of the AR.

The androgen receptor ligand-binding domain stabilizes DNA binding in living cells.

The androgen receptor (AR) is a member of the steroid receptor family, a group of transcription factors that activate steroid-regulated genes. Live cell studies of several steroid receptors have shown that the mobility of the liganded receptor is strongly reduced compared to the unliganded receptor. To investigate the nature of this reduced mobility, we generated Hep3B cells stably expressing green fluorescent protein (GFP)-AR at physiological levels. Computer-aided analysis of photobleaching experiments showed that in the presence of ligand on average one out of five ARs is immobilized, each individual AR being immobile for 1-2 min. This immobilization depended on DNA binding since GFP-ARs mutated in the DNA-binding domain were not immobilized. Interestingly, a truncated AR lacking the ligand-binding domain (LBD) displayed substantially shorter immobilizations, in the order of seconds, although its transcriptional activation function was stronger. Our data suggest the LBD has a role in maintaining the stability of AR-DNA complexes.